Mesenchymal Stromal Cells From Emphysematous Donors and Their Extracellular Vesicles Are Unable to Reverse Cardiorespiratory Dysfunction in Experimental Severe Emphysema

Although bone marrow-derived mesenchymal stromal cells (BM-MSCs) from patients with chronic obstructive pulmonary disease (COPD) appear to be phenotypically and functionally similar to BM-MSCs from healthy sources in vitro, the impact of COPD on MSC metabolism and mitochondrial function has not been evaluated. In this study, we aimed to comparatively characterize MSCs from healthy and emphysematous donors (H-MSCs and E-MSCs) in vitro and to assess the therapeutic potential of these MSCs and their extracellular vesicles (H-EVs and E-EVs) in an in vivo model of severe emphysema. For this purpose, C57BL/6 mice received intratracheal porcine pancreatic elastase once weekly for 4 weeks to induce emphysema; control animals received saline under the same protocol. Twenty-four hours after the last instillation, animals received saline, H-MSCs, E-MSCs, H-EVs, or E-EVs intravenously. In vitro characterization demonstrated that E-MSCs present downregulation of anti-inflammatory (TSG-6, VEGF, TGF-β, and HGF) and anti-oxidant (CAT, SOD, Nrf2, and GSH) genes, and their EVs had larger median diameter and lower average concentration. Compared with H-MSC, E-MSC mitochondria also exhibited a higher respiration rate, were morphologically elongated, expressed less dynamin-related protein-1, and produced more superoxide. When co-cultured with alveolar macrophages, both H-MSCs and E-MSCs induced an increase in iNOS and arginase-1 levels, but only H-MSCs and their EVs were able to enhance IL-10 levels. In vivo, emphysematous mice treated with E-MSCs or E-EVs demonstrated no amelioration in cardiorespiratory dysfunction. On the other hand, H-EVs, but not H-MSCs, were able to reduce the neutrophil count, the mean linear intercept, and IL-1β and TGF-β levels in lung tissue, as well as reduce pulmonary arterial hypertension and increase the right ventricular area in a murine model of elastase-induced severe emphysema. In conclusion, E-MSCs and E-EVs were unable to reverse cardiorespiratory dysfunction, whereas H-EVs administration was associated with a reduction in cardiovascular and respiratory damage in experimental severe emphysema.

Download Full-text

Enhanced anti-inflammatory effects of mesenchymal stromal cells mediated by the transient ectopic expression of CXCR4 and IL10

Stem Cell Research & Therapy ◽

10.1186/s13287-021-02193-0 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Rosario Hervás-Salcedo ◽

María Fernández-García ◽

Miriam Hernando-Rodríguez ◽

Oscar Quintana-Bustamante ◽

Jose-Carlos Segovia ◽

...

Keyword(s):

Transient Expression ◽

Mesenchymal Stromal Cells ◽

Stromal Cells ◽

Therapeutic Potential ◽

Ectopic Expression ◽

In Vivo Studies ◽

Anti Inflammatory ◽

The Impact

Abstract Background Mesenchymal stromal cells (MSCs) constitute one of the cell types most frequently used in cell therapy. Although several studies have shown the efficacy of these cells to modulate inflammation in different animal models, the results obtained in human clinical trials have been more modest. Here, we aimed at improving the therapeutic properties of MSCs by inducing a transient expression of two molecules that could enhance two different properties of these cells. With the purpose of improving MSC migration towards inflamed sites, we induced a transient expression of the C-X-C chemokine receptor type 4 (CXCR4). Additionally, to augment the anti-inflammatory properties of MSCs, a transient expression of the anti-inflammatory cytokine, interleukin 10 (IL10), was also induced. Methods Human adipose tissue-derived MSCs were transfected with messenger RNAs carrying the codon-optimized versions of CXCR4 and/or IL10. mRNA-transfected MSCs were then studied, first to evaluate whether the characteristic phenotype of MSCs was modified. Additionally, in vitro and also in vivo studies in an LPS-induced inflamed pad model were conducted to evaluate the impact associated to the transient expression of CXCR4 and/or IL10 in MSCs. Results Transfection of MSCs with CXCR4 and/or IL10 mRNAs induced a transient expression of these molecules without modifying the characteristic phenotype of MSCs. In vitro studies then revealed that the ectopic expression of CXCR4 significantly enhanced the migration of MSCs towards SDF-1, while an increased immunosuppression was associated with the ectopic expression of IL10. Finally, in vivo experiments showed that the co-expression of CXCR4 and IL10 increased the homing of MSCs into inflamed pads and induced an enhanced anti-inflammatory effect, compared to wild-type MSCs. Conclusions Our results demonstrate that the transient co-expression of CXCR4 and IL10 enhances the therapeutic potential of MSCs in a local inflammation mouse model, suggesting that these mRNA-modified cells may constitute a new step in the development of more efficient cell therapies for the treatment of inflammatory diseases.

Download Full-text

Mesenchymal stromal cells: a novel therapy for the treatment of chronic obstructive pulmonary disease?

Thorax ◽

10.1136/thoraxjnl-2017-210672 ◽

2018 ◽

Vol 73 (6) ◽

pp. 565-574 ◽

Cited By ~ 32

Author(s):

Winifred Broekman ◽

Padmini P S J Khedoe ◽

Koen Schepers ◽

Helene Roelofs ◽

Jan Stolk ◽

...

Keyword(s):

Clinical Trials ◽

Mesenchymal Stromal Cells ◽

Stromal Cells ◽

Animal Studies ◽

Chronic Obstructive ◽

Tissue Destruction ◽

Novel Therapy ◽

Model Studies

COPD is characterised by tissue destruction and inflammation. Given the lack of curative treatments and the progressive nature of the disease, new treatments for COPD are highly relevant. In vitro cell culture and animal studies have demonstrated that mesenchymal stromal cells (MSCs) have the capacity to modify immune responses and to enhance tissue repair. These properties of MSCs provided a rationale to investigate their potential for treatment of a variety of diseases, including COPD. Preclinical models support the hypothesis that MSCs may have clinical efficacy in COPD. However, although clinical trials have demonstrated the safety of MSC treatment, thus far they have not provided evidence for MSC efficacy in the treatment of COPD. In this review, we discuss the rationale for MSC-based cell therapy in COPD, the main findings from in vitro and in vivo preclinical COPD model studies, clinical trials in patients with COPD and directions for further research.

Download Full-text

miR-4732-3p in Extracellular Vesicles From Mesenchymal Stromal Cells Is Cardioprotective During Myocardial Ischemia

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.734143 ◽

2021 ◽

Vol 9 ◽

Author(s):

Rafael Sánchez-Sánchez ◽

Marta Gómez-Ferrer ◽

Ignacio Reinal ◽

Marc Buigues ◽

Estela Villanueva-Bádenas ◽

...

Keyword(s):

Extracellular Vesicles ◽

Mesenchymal Stromal Cells ◽

Transforming Growth Factor Beta ◽

Stromal Cells ◽

Transforming Growth Factor ◽

Lentiviral Vector ◽

Glucose Deprivation ◽

Scar Tissue

Extracellular vesicles (EVs) derived from mesenchymal stromal cells (MSCs) are an emerging alternative to cell-based therapies to treat many diseases. However, the complexity of producing homogeneous populations of EVs in sufficient amount hampers their clinical use. To address these limitations, we immortalized dental pulp-derived MSC using a human telomerase lentiviral vector and investigated the cardioprotective potential of a hypoxia-regulated EV-derived cargo microRNA, miR-4732-3p. We tested the compared the capacity of a synthetic miR-4732-3p mimic with EVs to confer protection to cardiomyocytes, fibroblasts and endothelial cells against oxygen-glucose deprivation (OGD). Results showed that OGD-induced cardiomyocytes treated with either EVs or miR-4732-3p showed prolonged spontaneous beating, lowered ROS levels, and less apoptosis. Transfection of the miR-4732-3p mimic was more effective than EVs in stimulating angiogenesis in vitro and in vivo and in reducing fibroblast differentiation upon transforming growth factor beta treatment. Finally, the miR-4732-3p mimic reduced scar tissue and preserved cardiac function when transplanted intramyocardially in infarcted nude rats. Overall, these results indicate that miR-4732-3p is regulated by hypoxia and exerts cardioprotective actions against ischemic insult, with potential application in cell-free-based therapeutic strategies.

Download Full-text

In Vitro Study of Extracellular Vesicles Migration in Cartilage-Derived Osteoarthritis Samples Using Real-Time Quantitative Multimodal Nonlinear Optics Imaging

Pharmaceutics ◽

10.3390/pharmaceutics12080734 ◽

2020 ◽

Vol 12 (8) ◽

pp. 734

Author(s):

Leonardo Mortati ◽

Laura de Girolamo ◽

Carlotta Perucca Orfei ◽

Marco Viganò ◽

Marco Brayda-Bruno ◽

...

Keyword(s):

Real Time ◽

Extracellular Vesicles ◽

Mesenchymal Stromal Cells ◽

Stromal Cells ◽

Second Harmonic ◽

Time Lapse ◽

Cartilage Explants ◽

Innovative Technology

Mesenchymal stromal cells (MSCs)-derived extracellular vesicles (EVs) are promising therapeutic nano-carriers for the treatment of osteoarthritis (OA). The assessment of their uptake in tissues is mandatory but, to date, available technology does not allow to track and quantify incorporation in real-time. To fill this knowledge gap, the present study was intended to develop an innovative technology to determine kinetics of fluorescent MSC-EV uptake by means of time-lapse quantitative microscopy techniques. Adipose-derived mesenchymal stromal cells (ASCs)-EVs were fluorescently labeled and tracked during their uptake into chondrocytes micromasses or cartilage explants, both derived from OA patients. Immunofluorescence and time-lapse coherent anti-Stokes Raman scattering, second harmonic generation and two-photon excited fluorescence were used to follow and quantify incorporation. EVs penetration appeared quickly after few minutes and reached 30–40 μm depth after 5 h in both explants and micromasses. In explants, uptake was slightly faster, with EVs signal overlapping both extracellular matrix and chondrocytes, whereas in micromasses a more homogenous diffusion was observed. The finding of this study demonstrates that this innovative technology is a powerful tool to monitor EVs migration in tissues characterized by a complex extracellular network, and to obtain data resembling in vivo conditions.

Download Full-text

Extracellular Vesicles Isolated from Mesenchymal Stromal Cells Primed with Hypoxia: Novel strategy in Regenerative Medicine

Current Stem Cell Research & Therapy ◽

10.2174/1574888x15999200918110638 ◽

2020 ◽

Vol 15 ◽

Author(s):

Shalmali Pendse ◽

Vaijayanti Kale ◽

Anuradha Vaidya

Keyword(s):

Extracellular Vesicles ◽

Mesenchymal Stromal Cells ◽

Stromal Cells ◽

Therapeutic Potential ◽

Ex Vivo ◽

Cell Types ◽

Molecular Profile ◽

Cell Contact ◽

Hematopoietic Stem

: Mesenchymal stromal cells (MSCs) regulate other cell types through a strong paracrine component called the secretome, comprising of several bioactive entities. The composition of the MSCs’ secretome is dependent upon the microenvironment in which they thrive, and hence, it could be altered by pre-conditioning the MSCs during in vitro culture. The primary aim of this review is to discuss various strategies that are being used for pre-conditioning of MSCs, also known as “priming of MSCs”, in the context of improving their therapeutic potential. Several studies have underscored the importance of extracellular vesicles (EVs) derived from primed MSCs in improving their efficacy in the treatment of various diseases. We have previously shown that co-culturing hematopoietic stem cells (HSCs) with hypoxiaprimed MSCs improves their engraftment potential. Now the question we pose is would priming of MSCs with hypoxiafavorably alter theirsecretome and would this altered secretome work as effectively as the cell to cell contact did? Here we review the current strategies of using the secretome, specifically the EVs (microvesicles and exosomes), collected from the primed MSCs with the intention of expanding HSCs ex vivo. We speculate that an effective priming of MSCs in vitrocould modulate the molecular profile of their secretome, which could eventually be used as a cell-free biologic in clinical settings.

Download Full-text

The Impact of Various Culture Conditions on Human Mesenchymal Stromal Cells Metabolism

Stem Cells International ◽

10.1155/2021/6659244 ◽

2021 ◽

Vol 2021 ◽

pp. 1-15

Author(s):

Pavla Tonarova ◽

Katerina Lochovska ◽

Robert Pytlik ◽

Marie Hubalek Kalbacova

Keyword(s):

Human Serum ◽

Mesenchymal Stromal Cells ◽

Stromal Cells ◽

Reciprocal Relationship ◽

Heat Inactivation ◽

High Quality ◽

Native Form ◽

The Impact

In vitro and in vivo analyses are closely connected, and the reciprocal relationship between the two comprises a key assumption with concern to the conducting of meaningful research. The primary purpose of in vitro analysis is to provide a solid background for in vivo and clinical study purposes. The fields of cell therapy, tissue engineering, and regenerative medicine depend upon the high quality and appropriate degree of the expansion of mesenchymal stromal cells (MSCs) under low-risk and well-defined conditions. Hence, it is necessary to determine suitable alternatives to fetal bovine serum (FBS—the laboratory gold standard) that comply with all the relevant clinical requirements and that provide the appropriate quantity of high-quality cells while preserving the required properties. Human serum (autologous and allogeneic) and blood platelet lysates and releasates are currently considered to offer promising and relatively well-accessible MSC cultivation alternatives. Our study compared the effect of heat-inactivated FBS on MSC metabolism as compared to its native form (both are used as the standard in laboratory practice) and to potential alternatives with concern to clinical application—human serum (allogeneic and autologous) or platelet releasate (PR-SRGF). The influence of the origin of the serum (fetal versus adult) was also determined. The results revealed the key impact of the heat inactivation of FBS on MSCs and the effectiveness of human sera and platelet releasates with respect to MSC behaviour (metabolic activity, proliferation, morphology, and cytokine production).

Download Full-text

Exofucosylation of Adipose Mesenchymal Stromal Cells Alters Their Secretome Profile

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2020.584074 ◽

2020 ◽

Vol 8 ◽

Author(s):

David García-Bernal ◽

Mariano García-Arranz ◽

Ana I. García-Guillén ◽

Ana M. García-Hernández ◽

Miguel Blanquer ◽

...

Keyword(s):

Mesenchymal Stromal Cells ◽

Stromal Cells ◽

Culture Conditions ◽

Peripheral Tissues ◽

Standard Culture ◽

Anti Inflammatory ◽

Selectin Binding ◽

The Impact

Mesenchymal stromal cells (MSCs) constitute the cell type more frequently used in many regenerative medicine approaches due to their exclusive immunomodulatory properties, and they have been reported to mediate profound immunomodulatory effects in vivo. Nevertheless, MSCs do not express essential adhesion molecules actively involved in cell migration, a phenotypic feature that hampers their ability to home inflamed tissues following intravenous administration. In this study, we investigated whether modification by fucosylation of murine AdMSCs (mAdMSCs) creates Hematopoietic Cell E-/L-selectin Ligand, the E-selectin-binding CD44 glycoform. This cell surface glycan modification of CD44 has previously shown in preclinical studies to favor trafficking of mAdMSCs to inflamed or injured peripheral tissues. We analyzed the impact that exofucosylation could have in other innate phenotypic and functional properties of MSCs. Compared to unmodified counterparts, fucosylated mAdMSCs demonstrated higher in vitro migration, an altered secretome pattern, including increased expression and secretion of anti-inflammatory molecules, and a higher capacity to inhibit mitogen-stimulated splenocyte proliferation under standard culture conditions. Together, these findings indicate that exofucosylation could represent a suitable cell engineering strategy, not only to facilitate the in vivo MSC colonization of damaged tissues after systemic administration, but also to convert MSCs in a more potent immunomodulatory/anti-inflammatory cell therapy-based product for the treatment of a variety of autoimmune, inflammatory, and degenerative diseases.

Download Full-text

Renal Regenerative Potential of Extracellular Vesicles Derived from miRNA-Engineered Mesenchymal Stromal Cells

International Journal of Molecular Sciences ◽

10.3390/ijms20102381 ◽

2019 ◽

Vol 20 (10) ◽

pp. 2381 ◽

Cited By ~ 11

Author(s):

Marta Tapparo ◽

Stefania Bruno ◽

Federica Collino ◽

Gabriele Togliatto ◽

Maria Chiara Deregibus ◽

...

Keyword(s):

Extracellular Vesicles ◽

Mesenchymal Stromal Cells ◽

Stromal Cells ◽

Therapeutic Interventions ◽

In Vivo Models ◽

Regenerative Effect ◽

In Vivo Experiments ◽

Renal Regeneration

Extracellular vesicles (EVs) derived from mesenchymal stromal cells (MSCs) possess pro-regenerative potential in different animal models with renal injury. EVs contain different molecules, including proteins, lipids and nucleic acids. Among the shuttled molecules, miRNAs have a relevant role in the pro-regenerative effects of EVs and are a promising target for therapeutic interventions. The aim of this study was to increase the content of specific miRNAs in EVs that are known to be involved in the pro-regenerative effect of EVs, and to assess the capacity of modified EVs to contribute to renal regeneration in in vivo models with acute kidney injuries. To this purpose, MSCs were transiently transfected with specific miRNA mimics by electroporation. Molecular analyses showed that, after transfection, MSCs and derived EVs were efficiently enriched in the selected miRNAs. In vitro and in vivo experiments indicated that EVs engineered with miRNAs maintained their pro-regenerative effects. Of relevance, engineered EVs were more effective than EVs derived from naïve MSCs when used at suboptimal doses. This suggests the potential use of a low amount of EVs (82.5 × 106) to obtain the renal regenerative effect.

Download Full-text

POS0374 SENESCENCE DID NOT ALTER THE CHONDROPROTECTIVE EFFECT OF EXTRACELLULAR VESICLES FROM MESENCHYMAL STROMAL CELLS

Annals of the Rheumatic Diseases ◽

10.1136/annrheumdis-2021-eular.2401 ◽

2021 ◽

Vol 80 (Suppl 1) ◽

pp. 417.2-418

Author(s):

J. Boulestreau ◽

M. Maumus ◽

P. Rozier ◽

C. Jorgensen ◽

D. Noel

Keyword(s):

Extracellular Vesicles ◽

Mesenchymal Stromal Cells ◽

Stromal Cells ◽

Total Protein Content ◽

Type Iii Collagen ◽

Γh2ax Foci ◽

Oa Chondrocytes ◽

The Impact ◽

Chondroprotective Effect

Background:Age is the most important risk factor in degenerative osteoarthritis (OA) and is associated with the accumulation of senescent cells that contribute to functional decline of joint. We previously demonstrated that extracellular vesicles (EVs) from mesenchymal stromal cells (MSCs) largely mediate the therapeutic effect of parental cells in OA.Objectives:Here, we assessed the impact of senescence on the characteristics of EVs from adipose tissue-derived MSCs (ASC-EVs) and their properties in an in vitro model of OAMethods:ASCs were induced to senescence using 25µM etoposide for 24 hours. Senescence was assessed by quantifying proliferation rate, SA-βGal activity, nuclear γH2AX foci number, phalloidin staining and expression of cyclin dependent kinase inhibitors (CDKI) (RT-qPCR). ASC-EVs were isolated by differential ultracentrifugation and characterized by size, concentration, total protein content, structure (cryo-TEM) and immunophenotype. In vitro OA model used chondrocytes isolated from OA patients, which were stimulated with IL1β for 48h before culture with ASCs or ASC-EVs for 7 days. Expression of chondrocytic and inflammatory markers was quantified by RT-qPCR and SASP factors were quantified by ELISA in supernatants.Results:Senescence-induced ASCs experienced growth arrest and increase of SA-βGal staining, of p21 CDKI expression, of nuclear γH2AX foci, of stress fibers and of several SASP factors (IL6, IL8, MMP3) confirming the expression of main senescence features. Senescent ASCs produced 4-fold more EVs than healthy ASCs and senescent ASC-EVs were larger. In vitro, both healthy and senescent ASCs decreased fibrotic markers (type III COLLAGEN), catabolic and hypertrophic markers (MMP3, MMP13, AP) and increased COX2 expression in OA chondrocytes. By contrast, healthy ASCs decreased the expression of IL6 while senescent ASCs highly increased IL6. Looking at the role of ASC-EVs on OA chondrocytes, we found out that both healthy and senescent ASC-EVs were able to increase the expression of AGG and type II COLLAGEN while they decreased the expression of MMP13, AP, type X COLLAGEN, HMOX1 and IL6. Finally, healthy and senescent ASC-EVs decreased the number of SA-βGal positive chondrocytes but did not impact the expression of p21 in IL1β-induced chondrocytes.Conclusion:Our results indicated a chondroprotective effect of ASC-EVs, independently of the senescent state of parental cells and suggested that EVs might act through different mechanisms than ASCs, which warrants further investigationDisclosure of Interests:Jérémy Boulestreau: None declared, Marie Maumus Employee of: Bauerfeind France, Pauline Rozier: None declared, Christian Jorgensen Shareholder of: Medxcell sciences, Consultant of: Medxcell sciences, Daniele Noel Shareholder of: Medxcell sciences, Consultant of: Medxcell sciences

Download Full-text

Extracellular Vesicles from Mesenchymal Stromal Cells for the Treatment of Inflammation-Related Conditions

International Journal of Molecular Sciences ◽

10.3390/ijms22063023 ◽

2021 ◽

Vol 22 (6) ◽

pp. 3023

Author(s):

Sean T. Ryan ◽

Elham Hosseini-Beheshti ◽

Dinara Afrose ◽

Xianting Ding ◽

Binbin Xia ◽

...

Keyword(s):

Extracellular Vesicles ◽

Mesenchymal Stromal Cells ◽

Stromal Cells ◽

Animal Studies ◽

Therapeutic Potential ◽

Early Stage ◽

Small Animal ◽

Therapeutic Effects ◽

Anti Inflammatory

Over the past two decades, mesenchymal stromal cells (MSCs) have demonstrated great potential in the treatment of inflammation-related conditions. Numerous early stage clinical trials have suggested that this treatment strategy has potential to lead to significant improvements in clinical outcomes. While promising, there remain substantial regulatory hurdles, safety concerns, and logistical issues that need to be addressed before cell-based treatments can have widespread clinical impact. These drawbacks, along with research aimed at elucidating the mechanisms by which MSCs exert their therapeutic effects, have inspired the development of extracellular vesicles (EVs) as anti-inflammatory therapeutic agents. The use of MSC-derived EVs for treating inflammation-related conditions has shown therapeutic potential in both in vitro and small animal studies. This review will explore the current research landscape pertaining to the use of MSC-derived EVs as anti-inflammatory and pro-regenerative agents in a range of inflammation-related conditions: osteoarthritis, rheumatoid arthritis, Alzheimer’s disease, cardiovascular disease, and preeclampsia. Along with this, the mechanisms by which MSC-derived EVs exert their beneficial effects on the damaged or degenerative tissues will be reviewed, giving insight into their therapeutic potential. Challenges and future perspectives on the use of MSC-derived EVs for the treatment of inflammation-related conditions will be discussed.

Download Full-text